1. Field of the Invention
The present invention relates to a process for preparing O-acetylserine, L-cysteine and sulfur-containing compounds derived therefrom.
2. The Prior Art
L-cysteine and its derivatives are employed in the pharmaceutical sphere (treatment of bronchial diseases), in the cosmetics sector (as constituents of hair shampoos and permanent wave lotions) and in the foodstuffs sphere (as antioxidants, as flavor enhancers and as adjuvants in the working of the dough). L-cysteine has hitherto been obtained by extraction from keratin-containing material, such as hair, bristles, horns, hooves and feathers, or by the enzymic transformation of precursors. An overproduction of L-cysteine by microorganisms is very desirable since L-cysteine is not only an economically interesting compound but, in addition, as is evident from FIGS. 1-3, constitutes an important intermediate in the synthesis of glutathione, methionine and biotin.
In all organisms, L-cysteine occupies a key position in sulfur metabolism and is used in the synthesis of proteins, glutathione, biotin, methionine and other sulfur-containing metabolites. Furthermore, L-cysteine acts as a precursor in the biosynthesis of coenzyme A; in addition to this, L-cysteine can readily be oxidized to cystine. A close connection exists between the biosynthesis of L-cysteine and that of other amino acids such as L-serine, glycine and L-methionine.
The synthesis of L-cysteine (FIG. 4) has been investigated in detail in prokaryotes, in particular bacteria (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965; Kredich, N. M., 1987, Biosynthesis of Cysteine. In: Neidhardt F. C., Ingraham, J. L., Magasanik, B., Low. K. B., Schaechter, M., Umbarger, H. E. (eds) Escherichia coli and Salmonella typhimurium: cellular and molecular biology, Vol. 1. American Society for Microbiology, Washington D.C., 419-428). The key reaction consists of the transfer of an acetyl group to serine in order to produce O-acetylserine 1), followed by the substitution of the acetyl group by the SH group, resulting in the synthesis of L-cysteine 2).
1) L-serine+acetyl-coenzyme A.fwdarw.O-acetylserine+coenzyme A
2) O-acetylserine+H.sub.2 S.fwdarw.L-cysteine+acetate
In microorganisms and in plants, O-acetylserine, and not serine, acts as the immediate precursor of the carbon skeleton of L-cysteine (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965). The reaction in which the acetyl group is transferred in order to produce an activated form of L-serine is catalyzed by the serine acetyltransferase (EC 2.3.1.30) which is encoded by the cysE gene and is subject to strict control by the end product L-cysteine. The gene for serine acetyltransferase has already been cloned and the amino acid sequence which is deduced from the DNA sequence is known (Denk, D. and Bock, A. 1987, J. Gen. Microbiol. 133: 515-525).
The formation of L-cysteine itself is catalyzed by two O-acetylserine sulfhydrylase isoenzymes (EC 4.2.99.8), encoded by the genes cysK (O-acetylserine sulfhydrylase A) and cysM (O-acetylserine sulfhydrylase B), a reaction in which O-acetylserine functions as a .beta.-alanyl donor and H.sub.2 S as a .beta.-alanyl acceptor (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965), with the O-acetylserine sulfhydrylase A making the major contribution to the cysteine synthesis. In addition, O-acetylserine sulfhydrylase B (cysM) is able to utilize thiosulfate as a sulfur source (Sirko, A. et al., 1987, J. Gen. Microbiol. 133: 2719-2725). The O-acetylserine sulfhydrylase B catalyzes the reaction between O-acetylserine and thiosulfate to form S-sulfocysteine, which can then be converted to cysteine (Nakamura, T., et al, 1983, J. Bacteriol. 156, 656-662).
The end product inhibition by L-cysteine of the wild-type form of serine acetyltransferase is a physiologically important factor in the kinetic regulation of cysteine biosynthesis (Kredich, N. M. 1971, J. Biol. Chem. 246, 3474-3484; Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241, 4955-4965). The activity of the wild-type form of serine acetyltransferase is inhibited by cysteine. This inhibition has been investigated kinetically and was found to have a competitive character. An inhibitor constant K.sub.i =1.1.times.10.sup.-6 M was determined in the presence of 0.1 mM acetyl-coenzyme A and 1 mM L-serine (Kredich, N. M. 1971 and Tomkins G. M. 1966, J. Biol. Chem. 241, 4955-4965).
An example is known from the literature of it being possible to isolate a cysteine-prototrophic revertant, whose serine acetyltransferase activity exhibits an end product inhibition by L-cysteine which is only weakly pronounced due to an amino acid substitution in the coding region, by chemically mutagenizing a cysteine-auxotrophic strain with ethyl methanesulfonate (Denk, D., Bock, A., 1987, J. Gen. Microbiol. 133: 515-525). According to the literature reference mentioned, the feedback resistance of this mutant is elevated 10-fold. Consequently, the K.sub.i of this mutant is approx. 0.01 mM, when comparison is made with the wild-type form.